Antiretroviral APOBEC3 cytidine deaminases alter HIV-1 provirus integration site profiles

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile.


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Life sciences study design
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Sample size

Data exclusions
Integration site locations in the human genome were obtained from the GRCh37/hg19 database (https://hgdownload.soe.ucsc.edu/downloads.html). Source data are provided with this paper. The integration site sequencing data generated in this study have been deposited in the NCBI SRA database under accession codes SAMN31866157-SAMN31866258 [http://www.ncbi.nlm.nih.gov/bioproject/905178]. The source data generated in this study are provided in the Supplementary Information/Source Data file.
The parent study that had recruited participants was examining the risk of HIV-1 acquisition in women on hormonal contraceptives. Only women that self reported their sex were enrolled for this study after screening and informed consent. Not relevant to this study but all women underwent gynecological exam as part of standard of care and for the studies. Samples were obtained for future HIV research studies related to viral fitness that may impact disease progression in these women which pertains to this study. There was are also HIV-1 infected patients who are receiving antiretroviral treatment and tested for drug resistance as standard of care for which sex and gender is not screening tool or considered for normal patient care at the Joint Clinical Research Center.
None of the co-variate analyses or sociodemographic information was necessary to report in this study. Aside from Dr. Eric Arts, the other authors of this study were blinded to all clinical and participant information in the women enrolled in the clinical study. None of the authors including Dr. Arts has any patient identifiers or clinical information on the samples obtained for routine drug resistance testing as standard of care.
Samples were collected from the WHO, CAP, and NIH-VQA-accredited Center For AIDS Research (CFAR) Laboratory of the Joint Clinical Research Center (JCRC) in Kampala, Uganda. The JCRC is one of the first HIV treatment centers in the country to roll out ART and currently the only site licensed to provide INSTIs in the country. HIV-negative women of child-bearing age (18-35 years old) were recruited, volunteered (without compensation) as participants after counseling and signing a consent from approximately 2002 to 2007 in the Risk of HIV-1 Acquisition Study with Hormonal Contraceptive based on various inclusion and exclusion criteria. If a woman was diagnosed with HIV-1 during the parent study above, there was invitation to participate in an ancillary study to determine markers of disease progression, again under consent and following counseling. The patient database in the CFAR laboratory was used to access HIV-1 infected patient sample ID numbers only. A total of 93 previously frozen and banked PBMC samples from HIV-1 infected patients receiving routine treatment care at the JCRC some of which also came from the Hormonal Contraception and HIV-1 Genital Shedding and Disease Progression among Women with Primary HIV Infection (GS) study were randomly collected.
Ethical clearance was obtained from the IRBs at the JCRC and UHCMC/CWRU (EM-10-579 07 and 10-05-35) A total of 93 whole blood samples were collected from patients receiving routine treatment care at the JCRC in Uganda. We randomly selected as many Ugandan participants as possible in order to maximize the number of integration sites for analysis. Given that each participant hosted dozens of integrations sites, we were able to achieve robust statistical analysis from the 93 participants.
In our flow cytometry experiments with the dual-color HIV vector, we excluded cells not expressing either marker (csGFP-,mKO2-) which comprise uninfected or dead cells, cells containing defective proviruses, and/or cells containing proviruses latent for both csGFP and mKO2 expression. We were interested in analyzing latently infected cells but because we could not differentiate cells latent for both csGFP and mKO2 expression from uninfected cells or cells containing defective proviruses from this particular population of sorted cells, they were excluded from analysis. This was predetermined before the experiment took place. In our analysis of LTR sequences, to ensure that we extracted sequences with sufficient homology with the HIV-1 LTR, LTR sequences containing more than 5 mismatches with the reference Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. HIV-1 LTR sequence were filtered out and not included in the analysis. This was a predetermined quality control criteria.
All experimental infections were successfully replicated three independent times. We also performed infections at different concentrations of virus and different APOBEC3 concentrations (3 independent replicates) which all yielded results consistent with the conclusions of the study.
The patient database in the CFAR laboratory was used to identify patient samples that would contain HIV-1 infected cells. The 93 Ugandan participant samples were randomly selected from the cohort without bias for clinical status. Allocation of samples into different experimental groups was not relevant in this study because all samples were grouped into one experimental group of infected patient DNA.
Blinded DNA samples were processed and sequenced to determine integration site profiles. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

Antibodies used in Western blot and IP 1-Mouse anti-Human
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A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance Mouse anti-p24 (183-H12-5C) is used as a capture antibody in ELISA and primary antibody in Western blots at a concentration of 5ug/ ml. 31-90-25 p24 antibody is used as a primary antibody-biotin conjugated (10ug/ml). Mouse anti-FLAG is used at a concentration of (0.67ug/ml) and rabbit anti-beta-tubulin (0.25ug/ml).
Monoclonal anti-FLAG: The monoclonal antibody detects only the target protein band(s) on a Western blot from an E. coli, plant or mammalian crude cell lysate. The monoclonal antibody detects as little as 2 ng of target protein by dot blot. The Western blot is tested down to 10 ng. Validation: A Western blot was performed to demonstrate that the ANTI-FLAG M2, Affinity Purified antibody displays exquisite specificity for the epitope-tagged fusion protein. (https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/ product/documents/144/194/vol6_iss2_antiflag_m2.pdf.) Monoclonal mouse anti-Human Immunodeficiency Virus 1 (HIV-1): ARP-3537 is a monoclonal antibody to HIV-1 p24 .This antibody was produced in cell culture and purified by Protein G chromatography. It originates from a hybridoma. The hybridoma was created by immunizing a Balb/c mouse and fusing the resulting splenocytes with SP2/0 myeloma cells. This antibody is cross reactive with HIV-2 p24 and SIV p27 (Wehrly, K., & Chesebro, B. (1997). p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods,12 (4) None of the cell lines used were genetically authenticated. The cell lines were assessed morphologically.
Samples were run and analyzed on a BD FACSCelestra using BD FACSDiva (software v8.0.1) Post-acquisition analysis was performed on a separate computer using FlowJo (software v10.4.2) Initial cell populations were selected through the forward and side scatter plots by excluding debris and dead cells (smallFSC and SSC) followed by FSC-A/FSC-H gating to select singlet cells. 10,000-20,000 events were originally collected from which positively-gated cells showed 90-95% purity.